Solvent substitution as a probe of channel gating in Myxicola. Differential effects of D2O on some components of membrane conductance.

Careful examination of effects of solvent substitution on excitable membranes offers the theoretical possibility of identifying those aspects of the gating and translocation processes which are associated with significant changes in solvent order. Such information can then be used to develop or modify moire detailed models. We have examined the effects of heavy water substitution in Cs+-and K+-dialyzed Myxicola giant axons. At temperatures of 4-6 degrees C, the rates of Na+, K+, and Na+ inactivation during a maintained depolarization were all showed by approximately 50% in the presence of D2O. In contrast, the effects of solvent substitution on the time-course of prepulse inactivation and reactivation were much larger, with slowing averaging 160%. Studies at higher temperatures yielded Q10's for Na+ activation and K+ activation which were essentially comparable (0.72) and slightly but significantly smaller than that for inactivation during a maintained depolarization (0.84). In contrast, the Q10 for the D2O effect on prepulse inactivation was approximately 0.48. Heavy water substitution decrease Gk to a significantly greater extent than G(Na), while the decrease in the conductance of the Na+ channel caused by D2O was independent of whether the current-carrying species was Na+ or Li+. Sodium channel selectivity to the alkali metal cations and NH4+ was not changed by D2O substitution.     

The gap junction channel. Its aqueous nature as indicated by deuterium oxide effects.

The effects of temperature and solvent substitution with deuterium oxide (D2O) on axoplasmic (ga) and gap junctional (gj) conductances were examined in the earthworm septate median giant axon (MGA). The temperature coefficients (Q10) for ga and gj were 1.4 and 1.5, respectively, between 5 and 15 degrees C. Substitution with D2O rapidly reduced both ga and gj by 20% and increased the Q10's to 1.5 and 1.8, respectively. The reduction in ga upon substitution with D2O and with cooling in either solvent reflects the changes that occur in solvent viscosity, which indicates that ion mobility in axoplasm, as in free solution, is primarily governed by viscous properties of the solvent. The similar initial reduction observed for gj suggests that solvent occupies the gap junction channel volume and influences transjunctional ion mobility. With time there was a further reduction in gj at 20 degrees C and a larger Q10 in D2O. The enhanced effects of D2O on gj cannot be accounted for by solvent viscosity alone and may be due to an increased hydration of the channels and/or the transport ions and by isotope effects of hydrogen-deuterium exchange on the channel protein that reduce gj.     

Deuterium oxide and temperature effects on the properties of endplate channels at the frog neuromuscular junction

The effects of deuterium oxide (D2O) and temperature on the properties of endplate channels were studied in voltage-clamped muscle fibers from the frog Rana pipiens. Studies were performed at temperatures of 8, 12, 16, and 20 degrees C. The single channel conductance (gamma) and mean channel lifetime (tau) were calculated from fluctuation analysis of the acetylcholine-induced end-plate currents. The reversal potential was determined by interpolation of the acetylcholine-induced current-voltage relation. The mean reversal potential was slightly more negative in D2O Ringer's (-7.9 +/- 0.1 mV [+/- SEM]) compared with H2O Ringer's (-5.2 +/- 0.6 mV, P less than 0.01). The single channel conductance was decreased in D2O. This decrease was greater than could be accounted for by the increased viscosity of D2O solutions, and the amount of the decrease was greater at higher temperatures. For example, gamma was 38.4 +/- 1.3 pS (+/- SEM) in H2O Ringer's and 25.7 +/- 1.0 pS in D2O Ringer's for a holding potential of -70 mV at 12 degrees C. The mean channel lifetime was significantly shorter in D2O, and the effect was greater at lower temperatures. There was not a strong effect of solvent on the temperature dependence of gamma. On the other hand, the temperature dependence of the reciprocal mean channel lifetime, alpha (where alpha = 1/tau), was strongly dependent upon the solvent. The single channel conductances showed no demonstrable voltage dependence over the range of -90 to -50 mV in both solvents. The reciprocal mean channel lifetime showed a voltage dependence, which could be described by the relation alpha = B exp(AV). The slope A was not strongly affected by either temperature or the solvent. On the other hand, the intercept B was a strong function of temperature and was weakly dependent upon the solvent, with most values greater in D2O. The D2O effects on alpha were what would be expected if they were due to the properties of D2O as a solvent (solvent isotope effects), while the D2O effects on gamma must also include the exchange of D for H in the vicinity of the selectivity filter (primary and/or secondary kinetic isotope effects).     

Inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons

Inhibitory interactions between 5-HT subtype 3 (5-HT(3)) and P2X receptors were characterized using whole cell recording techniques. Currents induced by 5-HT (I(5-HT)) and ATP (I(ATP)) were blocked by tropisetron (or ondansetron) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, respectively. Currents induced by 5-HT + ATP (I(5-HT+ATP)) were only as large as the current induced by the most effective transmitter, revealing current occlusion. Occlusion was observed at membrane potentials of -60 and 0 mV (for inward currents), but it was not present at +40 mV (for outward currents). Kinetic and pharmacological properties of I(5-HT+ATP) indicate that they are carried through 5-HT(3) and P2X channels. Current occlusion occurred as fast as activation of I(5-HT) and I(ATP), was still present in the absence of Ca(2+) or Mg(2+), after adding staurosporine, genistein, K-252a, or N-ethylmaleimide to the pipette solution, after substituting ATP with proportional to, beta-methylene ATP or GTP with GTP-gamma-S in the pipette, and was observed at 35 degrees C, 23 degrees C, and 8 degrees C. These results are in agreement with a model that considers that 5-HT(3) and P2X channels are in functional clusters and that these channels might directly inhibit each other.     

Acylation of subtilisin A by aryl esters: contribution to rate limitation by a physical step preceding general acid-base catalysis

The specificity ratios kc/Km = k for subtilisin A catalyzed hydrolysis of five aryl esters of N-(methoxycarbonyl)-L-Phe (McPhe) were determined at pH 7.03 and its pD equivalent. The ratios are independent of the electronic properties of the leaving group substituent. Kinetic solvent isotope effects, Dk, increase from about 0.9 to 1.3 as leaving group ability decreases from p-nitrophenolate to p-methoxyphenolate. The k of N-(methoxycarbonyl)-L-phenylalanine p-nitrophenyl ester (NPE) with native enzyme exhibits a strong temperature dependence; delta H* = 87 +/- 3 kJ mol-1 and delta S* = 148 +/- 14 J K-1 mol-1 at 25 degrees C (H2O). The Dk with this substrate is 1.36 at 13.6 degrees C, declines to 0.89 at 25 degrees C, and then increases to 1.04 at 39.4 degrees C. Above neutral pH(D), with McPhe NPE as substrate, the dependence of k is for the dissociated form of a single base of pKapp = 7.38 +/- 0.03 in H2O and 7.67 +/- 0.03 in D2O. The pKapp values are apparently those of the uncomplexed native protein. By contrast, k of 3-phenylpropanoic acid (Prop) p-nitrophenyl ester exhibits a weaker temperature dependence; delta H* = 20 kJ mol-1 and delta S* = -90 J K-1 mol-1 (H2O) at 25 degrees C. The Dk are larger than those for McPhe NPE, decreasing from 1.99 at 20.5 degrees C to 1.74 at 46.1 degrees C. These results, combined with those of previous studies, are consistent with limitation of k by at least two processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of specific solvent rearrangement regions of an enzyme: NMR and ITC studies with aminoglycoside phosphotransferase(3')-IIIa

This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3')-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative DeltaH in H2O relative to D2O (DeltaDeltaH(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative DeltaCp values were observed for binding of aminoglycosides to APH. DeltaCp for the APH-neomycin complex was -1.6 kcal x mol(-1) x deg(-1). A break at 30 degrees C was observed in the APH-kanamycin complex yielding DeltaCp values of -0.7 kcal x mol(-1) x deg(-1) and -3.8 kcal x mol(-1) x deg(-1) below and above 30 degrees C, respectively. Neither the change in accessible surface area (DeltaASA) nor contributions from heats of ionization were sufficient to explain the large negative DeltaCp values. Most significantly, 15N-1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 degrees C only in the APH-kanamycin complex in spectra collected between 21 degrees C and 38 degrees C. These amino acids represent solvent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect DeltaCp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site.     

Structural aspects of the sarcoplasmic reticulum K+ channel revealed by gallamine block.

We have studied single-channel conductance fluctuations of K+ channels present in the sarcoplasmic reticulum (SR) membrane systems of rabbit cardiac and skeletal muscle. K+ conductance through the channels is reversibly blocked by gallamine. Conductance block occurs only from the trans side of the channel and is resolved as a smooth reduction in the open state conductance. At a fixed K+ concentration, conduction decreases with increasing gallamine concentration and the data can be fitted to a single-site inhibition scheme. The degree of block seen at a constant gallamine concentration decreases as K+ concentration is increased, indicating competition between gallamine and K+. Gallamine block is voltage dependent, the degree of block increasing with increasing negative holding potential. Quantitative analysis of block yields a zero voltage dissociation constant of 55.3 +/- 16 microM and an effective valence of block of 0.93 +/- 0.12. We conclude that gallamine blocks by interacting with a site or sites located at an electrical distance 30-35% into the voltage drop from the trans side of the channel. This site must have a cross-sectional area of at least 1.2 nm2. The results of this study have been used to modify and extend our view of the structure of the channel's conduction pathway.     

Stabilization of tubulin by deuterium oxide

Tubulin is an unstable protein when stored in solution and loses its ability to form microtubules rapidly. We have found that D2O stabilizes the protein against inactivation at both 4 and 37 degrees C. In H2O-based buffer, tubulin was completely inactivated after 40 h at 4 degrees C, but in buffer prepared in D2O, no activity was lost after 54 h. Tubulin was completely inactivated at 37 degrees C in 8 h in H2O buffer, but only 20% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slower rate in D2O. The deuterated solvent retarded an aggregation process that occurs during incubation at both temperatures. Inactivation in H2O buffer was partially reversed by transferring the protein to D2O buffer; however, aggregation was not reversed. The level of binding of BisANS, a probe of exposed hydrophobic sites in proteins, increases during the inactivation of tubulin. In D2O, the rate of this increase is slowed somewhat. We propose that D2O has its stabilizing effect on a conformational step or steps that involve the disruption of hydrophobic forces. The conformational change is followed by an aggregation process that cannot be reversed by D2O. As reported previously [Ito, T., and Sato, H. (1984) Biochim. Biophys. Acta 800, 21-27], we found that D2O stimulates the formation of microtubules from tubulin. We also observed that the products of assembly in D2O/8% DMSO consisted of a high percentage of ribbon structures and incompletely folded microtubules. When these polymers were disassembled and reassembled in H2O/8% DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO, both stimulators of tubulin assembly, leads to the rapid production of nuclei that lead to the formation of ribbon structures rather than microtubules.     

Oxygen consumption as related to the development of the extraembryonic membranes and cardiovascular system in the European pond turtle (Emys orbicularis) embryogenesis

The rate of oxygen consumption (V(O2)), embryo mass, distribution and mass of the chorioallantoic membrane (CAM), heart rate (HR), heart mass, and amnion rhythmic contractions (ARC) were studied in eggs of the European pond turtle (Emys orbicularis) incubated at 28 degrees C for 62.5+/-0.3 days. The V(O2) rapidly increased beginning from incubation day 19 (D19) to a maximum on D50 and then decreased until pipping. The rapid V(O2) rise was correlated with an increase in the CAM surface and mass, heart mass, and ARC amplitude, whereas the functional parameters such as HR and ARC frequency remained unchanged. The drop in V(O2) before pipping was accompanied by a decrease in HR, while the heart and CAM masses were almost constant. In the cases of short-term temperature deviations of +/-3 degrees C from 28 degrees C, changes in (O2) were significant until D50 and nonsignificant after that, the changes in ARC frequency and HR being significant at all stages studied. Thus, the developmental V(O2) changes were contributed mainly by the slow morphogenetic processes during D19-D50, whereas changes in functional parameters began to play a role at later stages. The response to temperature fluctuations was mediated by a rapid change in functional parameters at all these stages.     

Examination of microgravity effects on spontaneous Ca2+ oscillations in AtT20 pituitary cells using heavy water

Effects of heavy water (D2O) on various organisms have been extensively studied and a majority of D2O actions were generally ascribed to the viscosity (1.23 times of H2O) and a larger inter-molecule force of D2O that may eventually alternate molecular structure of various enzymes and ion channels. It is reported that chronic application of D2O induces toxic effects and the 35% substitution of whole body water with D2O induced fatal effects in the mouse. Mitosis of a fertile egg of sea urchin was completely inhibited by 75% D2O but the paused segmentation was recovered after rinse of D2O. In addition, we also observed that neuronal development of the Lymnaea stagnalis was reversibly inhibited by D2O (M. Sakakibara, unpublished data). However, mechanism of the toxicity of D2O and the effects of D2O on cellular events have not been fully understood. The spontaneous oscillation in cytosolic free Ca2+ concentration is one of the typical physiological events in living secretory cells. We previously demonstrated that the Ca2+ oscillations are regulated by voltage-sensitive Ca2+ channels (VSCC), Ca2+ ATPases, and Ca(2+)-induced Ca2+ release from intracellular stores. To analyze the site(s) of action of D2O in the living cellular systems, the present study examined effects of D2O on the Ca2+ mobilization and resting membrane potentials in AtT20 mouse pituitary cells.     

Thiazolium C(2)-proton exchange: structure-reactivity correlations and the pKa of thiamin C(2)-H revisited

Rate constants for C(2)-proton exchange from thiamin, N(1')-methylthiamin, and several 3-substituted-4-methylthiazolium ions catalyzed by D2O and deuterioxide ion were determined by 1H NMR at 30 degrees C and ionic strength 2.0 M. Values of pKa for the thiazolium ions, including thiamin itself, were found to be in the range pKa = 17-19; the pKa values for N(1')-protonated thiamin and free thiamin C(2)-H in H2O are 17.7 and 18.0, respectively. The pKa value for N(1')-protonated thiamin was calculated from the observed rate constant for the pD-independent reaction with D2O after correction for a secondary solvent deuterium isotope effect of kH2O/kD2O = 2.6. The pKa value for free thiamin was calculated from the rate constant for catalysis by OD- after correction by a factor of 3.3 = 8/2.4 for an 8-fold negative deviation of kOD from the Br?nsted plot of slope 1.0 for general base catalysis and a secondary solvent isotope effect of kOD/kOH = 2.4. Values of k-a = 2 X 10(10) and 3 X 10(9) M-1 s-1 were assumed for diffusion-controlled protonation of the C(2) ylide in the reverse direction by H3O+ and H2O, respectively. The Hammett rho I value for the exchange reaction catalyzed by deuterioxide ion or D2O is 8.4 +/- 0.2. There is no positive deviation of the rate constants for free or N(1')-substituted thiamin analogues in either Hammett correlation. This shows that the aminopyrimidinyl group does not provide significant intramolecular catalysis of nonenzymic C(2)-proton removal in the coenzyme.     

Modifications of sodium channel gating in Myxicola giant axons by deuterium oxide, temperature, and internal cations.

In dialyzed Myxicola axons substitution of heavy water (D2O) externally and internally slows both sodium and potassium kinetics and decreases the maximum conductances. Furthermore, this effect is strongly temperature dependent, the magnitude of the slowing produced by D2O substitution decreasing with increasing temperature over the range 3-14 degrees C with a Q10 of approximately 0.71. The relatively small magnitude of the D2O effect, combined with its strong temperature dependence, suggests that the rate limiting process producing a conducting channel involves appreciable local changes in solvent structure. Maximum conductances in the presence of D2O were decreased by approximately 30%, while the voltage dependences of both gNa and gK were not appreciably changed. In contrast to the effects of heavy water substitution on the ionic currents, membrane asymmetry currents were not altered by D2O, suggesting that gating charge movement may preceed by several steps the final transformation of the Na+ channel to a conducting state. In Myxicola axons the effect of temperature alone on asymmetry current kinetics can be well described via a simple temporal expansion equivalent to a Q10 of 2.2, which is somewhat less than the Q10 of GNa activation. The integral of membrane asymmetry current, representing maximum charge movement, is however not appreciably altered by temperature.     

Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two-dimensional NMR.

In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein-ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO-D2O mixtures was investigated by NMR methods. As in water, H-D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid- and base-catalyzed processes and negligible water catalysis. However, the base-catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is approximately 100-fold reduced relative to that in D2O. The solvent-dependent decrease in base-catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKW seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent-quenching method, melittin was exchanged in D2O (pH 3.2, 12 degrees C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.     

Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, deltaH(o), deltaS(o), and deltaC(o)p and evaluation of solvent isotope effects.

The stability of the N-terminal domain of the ribosomal protein L9, NTL9, from Bacillus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O. The basic thermodynamic parameters for the unfolding reaction, deltaH(o), deltaS(o), and deltaC(o)p, were determined by global analysis of temperature and denaturant effects on stability. The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs-Helmholtz equation to model changes in stability with temperature. The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations. NTL9 has a maximum stability of 3.78 +/- 0.25 kcal mol(-1) at 14 degrees C. DeltaH(o)(25 degrees C) for protein unfolding equals 9.9 +/- 0.7 kcal mol(-1) and TdeltaS(o)++(25 degrees C) equals 6.2 +/- 0.6 kcal mol(-1). DeltaC(o)p equals 0.53 +/- 0.06 kcal mol(-1) deg(-1). There is a small increase in stability when D2O is substituted for H2O. Based on the results from global analysis, NTL9 is 1.06 +/- 0.60 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 5.8 +/- 3.6 degrees C in D2O. Based on the results from individual denaturation experiments, NTL9 is 0.68 +/- 0.68 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 3.5 +/- 2.1 degrees C in D2O. Within experimental error there are no changes in deltaH(o) (25 degrees C) when D2O is substituted for H2O.     

Mechanism of O2 activation by cytochrome P450cam studied by isotope effects and transient state kinetics

The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.     

The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.     

Voltage and temperature dependence of single K+ channels isolated from canine cardiac sarcoplasmic reticulum.

The temperature and voltage dependence of gating and conductance of sarcoplasmic reticulum K+ channels (S-R K+) isolated from adult canine hearts were studied using the reconstituted bilayer technique. Fusion of vesicles from this preparation frequently resulted in the incorporation of a single channel. Only bilayers into which a single S-R K+ channel had fused were studied. The three conductance states of the channel, fully open (O2), substate conductance (O1), and closed (C) were studied as a function of voltage (-50 to +50 mV) and temperature (16 to 37 degrees C). Permeation through the O1 state showed the same temperature dependence as the O2 state corresponding to an enthalpy of permeation of 4.1-4.2 kcal/mol, which is similar to that for K+ diffusion through water. As expected, increased temperature increased the frequency of gating transitions and shortened the average dwell time spent in any conductance state. Over the range of 25 to 37 degrees C, the average dwell time spent in the O1, O2, and C states decreased by 44 +/- 11, 36 +/- 13, and 78 +/- 7% (n = 3 to 4 channels), respectively. The ratio of probabilities between the various conductance states was not strongly temperature sensitive. Analysis of the voltage dependence of this channel was carried out at 37 degrees C and revealed that the dwell times of the O1 and O2 states were voltage insensitive and the probability ratio (PO2:PO1) was approximately 7 and was voltage insensitive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occluded calcium sites in soluble sarcoplasmic reticulum Ca2+-ATPase

Rabbit muscle sarcoplasmic reticulum Ca2+-ATPase has been shown to bind gadolinium ion (Gd3+) at two high affinity Ca2+ sites (Stephens, E. M., and Grisham, C. M. (1979) Biochemistry 18, 4876-4885). Gd3+ bound at these sites exhibits an unusually long electron spin relaxation time, consistent with occlusion of these sites and reduced contact with solvent H2O. In this report, the nature of the Gd3+ sites was examined in preparations of the enzyme solubilized with the detergent C12E8. The frequency dependence of water proton relaxation in solutions containing the solubilized Ca2+-ATPase yields dipolar correlation times, tau c, for the 1H-Gd3+ interaction of 1.04 X 10(-9) s for Gd3+ bound at site 1 and 1.98 X 10(-9) s for Gd3+ bound at site 2. The correlation time itself is frequency dependent below 30 MHz, indicating that the correlation time is dominated by the electron spin relaxation time of bound Gd3+. The long values of the correlation time found in the present study are consistent with a poor accessibility of these Gd3+ sites (particularly site 2) to solvent water molecules. Analytical ultracentrifugation and molecular sieve high performance liquid chromatography indicated that the active fraction of the soluble Ca2+-ATPase was monomeric. Thus occlusion of the Ca2+ sites in this enzyme is largely dependent on the tertiary structure of the monomeric ATPase and does not appear to depend on multimeric membrane structures.     

Temperature dependence of Ca2+-activated K+ currents in the membrane of human erythrocytes

The currents through single Ca2+-activated K+ channels were studied in excised inside-out membrane patches of human erythrocytes. The effects of temperature on single-channel conductance, on channel gating and on activation by Ca2+ were investigated in the temperature range from 0 up to 47 degrees C. The single-channel conductance shows a continuous increase with increasing temperature; an Arrhenius plot of the conductance gives the activation energy of 29.6 +/- 0.4 kJ/mol. Reducing the temperature alters channel-gating kinetics which results in a significant increase of the probability of the channel being open (Po). The calcium dependence of Po is affected by temperature in different ways; the threshold concentration for activation by Ca2+ is not changed, the Ca2+ concentration of half-maximal channel activation is reduced from 2.1 mumol/l at 20 degrees C to 0.3 mumol/l at 0 degrees C, the saturation level of the dependence is reduced for temperatures higher then about 30 degrees C. The relevance of the obtained data for the interpretation of the results known from flux experiments on cells in suspensions is discussed.     

Thermodynamic study of protein dynamic structure in the oxygen binding reaction of myoglobin

We examined the flash photolysis of oxy complexes of sperm whale myoglobin (Mb) on the nanosecond time scale at ambient temperatures. In this time range, we can observe the geminate reaction of Mb with the O2 ligand existing in the protein matrix after the photodissociation from the heme iron. We found that the fraction of the geminate component to the total O2 photodissociation exhibited temperature dependences. The geminate fraction decreased with rising temperature, indicating that the protein fluctuation is enhanced at high temperature because of thermal agitation. However, the temperature-dependent behavior showed a break at 20 degrees C. Concerning the geminate O2 escaping reaction from the protein matrix to the solvent region, the activation energy above 20 degrees C (0.4 +/- 0.4 kcal/mol) is significantly lower than that below 20 degrees C (5.1 +/- 0.4 kcal/mol). Thermodynamic analysis on the basis of the transition state theory indicated that the O2 escaping reaction above 20 degrees C is entropy dominated whereas that below 20 degrees C is enthalpy dominated. The results were qualitatively compatible with the theoretical prediction by J. Kottalam and D. A. Case [1988) J. Am. Chem. Soc. 110, 7690-7697). Comparing the kinetic and thermodynamic process of the O2 geminate reaction among several Mbs, we concluded that the geminate O2 reaction with Mb is governed by the dynamic motion of the protein which is sensitively controlled by the static interaction of the heme moiety with the surroundings.